M. tuberculosis (Mtb), a leading cause of death in people with HIV/AIDS and a bioterrorism threat, persists in macrophages, where reactive nitrogen intermediates (RNI) from inducible nitric oxide synthase (iNOS) and reactive oxygen intermediates (ROI) from phagocyte oxidase are opposed by Mtb's RNI/ROI resistance mechanisms. RNI kill in part via intrabacterial conversion into peroxynitrite (OONO-) (PN). We have discovered a biochemically novel PN reductase/peroxidase (PNRP) in Mtb, comprised of 4 proteins: AhpC, AhpD, succinyl-coenzyme A acyltransferase (SucB) and lipoamide dehydrogenase (Lpd). Lpd uses its flavin to transfer electrons from NADH to the lipoamide cofactor covalently coupled to SucB. SucB's lipoamide transfers electrons to a Trx-like active site we have identified by X-ray crystallography within a novel fold in AhpD. AhpD's Cys130 and Cys133 cooperate to transfer electrons to the disulfide in oxidized AhpC. The AhpC disulfide arises after formation of a sulfenic acid intermediate during reduction of PN to nitrite or peroxides to the alcohol. Besides participating in this antioxidant pathway, Lpd and SucB appear to serve as shared, perhaps essential components of all of Mtb's alpha-keto acid dehydrogenase complexes (pyruvate, alpha-ketoglutarate and branched chain ketoacid dehydrogenases), thereby supplying much of the acetyl coenzyme A arising from endogenous sources. Acetyl CoA is essential for the glyoxylate shunt that sustains bacillary persistence and for fatty acid synthesis required by Mtb to build its cell wall. Thus, Mtb Lpd and SucB stand at a crossroads between antioxidant defense and intermediary metabolism. In the work proposed here, we will knock out Lpd, SucB and AhpD and study the impact on growth of Mtb in vitro; its sensitivity to RNI, ROI and control stresses; its growth in macrophages that are wild type, deficient in iNOS, deficient in phagocyte oxidase or deficient in both enzymes; and its growth in these four strains of mice. We will solve the crystal structures of Lpd and SucB and identify chemical inhibitors of the three enzymes by screening chemical libraries and by directed synthesis. This work will characterize Lpd, SucB and AhpD as potential targets for interventions that may cripple the growth of Mtb while sensitizing the organism to RNI, ROI and chemotherapy for improved prophylaxis and treatment in normal and immunocompromised hosts.